In battling existing and newly emerging viral infections and cancer, nucleoside analogues (NAs) represent a prominent and highly potent weapon in the medicinal arsenal. Administered as membrane-penetrating nucleosidic prodrugs, the compounds require intracellular activation by deoxynucleoside kinases (dNKs) and deoxynucleotide kinases (dNPKs) into their bioactive triphosphate anabolites. In recent years, dNKs and dNPKs have taken on a key role in the quest for new and more potent antiviral and anticancer drugs. Emerging resistance to NAs upon long-term exposure, accumulation of cytotoxic intermediates, and failure of a large percentage of new prodrugs in vivo has been linked to problems associated with the phosphorylation cascade. The proposed research program focuses on the engineering of mammalian and bacterial dNKs and dNPKs, tailoring catalysts for optimal specificity toward NAs to maximize prodrug efficiency upon codelivery in cancer and antiviral therapy. Employing a novel, homology- independent combinatorial technique named SCRATCHY enables us to produce vast numbers of hybrid constructs from parents with high sequence diversity as found in the dNK/dNPK family. Functional hybrid candidates are subsequently selected by genetic selection and high-throughput screening. We will 1) Demonstrate that protein fragment swapping between structure-homologous proteins can generate hybrid enzymes that exhibit desired function, 2) Validate the performance of hybrid kinases through co-administration with prodrugs in cell-based assays, and 3) Develop a framework to study the structure-function relationship of dNKs and dNPKs. The specific aims of this work are: 1) To identify engineered dNKs with improved NA specificity, 2) To generate novel catalysts that perform multistep phosphorylation, 3) To improve the versatility of structure-based protein engineering, and 4) To evaluate functional hybrid enzymes on established and novel NAs in vitro and in vivo. The results from these studies will have far-reaching implications on viral disease and cancer treatment, affecting existing prodrugs as well as potential drug candidates by facilitating their phosphorylation independent of cellular dNK activation. Finally, the structural diversity of the hybrid proteins will be a rich source for fundamental structure-function studies.